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High-performance binary protein interaction screening in a microfluidic format.

ABSTRACT: The standard procedure to increase microfluidic chip performance is to grow the number of parallel test systems on the chip. This process is accompanied by miniaturizing biochemical workflows and micromechanical elements, which is often a major challenge for both engineering fields. In this work, we show that it is possible to substantially increase the runtime performance of a microfluidic affinity assay for protein interactions by simultaneously engineering fluid logics and assay chemistry. For this, synergistic effects between the micro- and chemical architecture of the chip are exploited. The presented strategy of reducing the runtime rather than size and volume of the mechanical elements and biological reagent compartments will, in general, be of importance for future analytical test systems on microfluidic chips to overcome performance barriers.

SUBMITTER: Meier M

PROVIDER: S-EPMC3533494 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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High-performance binary protein interaction screening in a microfluidic format.

Meier Matthias M Sit Rene R Pan Wenying W Quake Stephen R SR

Analytical chemistry 20121023 21

The standard procedure to increase microfluidic chip performance is to grow the number of parallel test systems on the chip. This process is accompanied by miniaturizing biochemical workflows and micromechanical elements, which is often a major challenge for both engineering fields. In this work, we show that it is possible to substantially increase the runtime performance of a microfluidic affinity assay for protein interactions by simultaneously engineering fluid logics and assay chemistry. Fo ...[more]

PMID: 23051662

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Project description:Functional imaging can provide a level of quantification that is not possible in what might be termed traditional high-content screening. This is due to the fact that the current state-of-the-art high-content screening systems take the approach of scaling-up single cell assays, and are therefore based on essentially pictorial measures as assay indicators. Such phenotypic analyses have become extremely sophisticated, advancing screening enormously, but this approach can still be somewhat subjective. We describe the development, and validation, of a prototype high-content screening platform that combines steady-state fluorescence anisotropy imaging with fluorescence lifetime imaging (FLIM). This functional approach allows objective, quantitative screening of small molecule libraries in protein-protein interaction assays. We discuss the development of the instrumentation, the process by which information on fluorescence resonance energy transfer (FRET) can be extracted from wide-field, acceptor fluorescence anisotropy imaging and cross-checking of this modality using lifetime imaging by time-correlated single-photon counting. Imaging of cells expressing protein constructs where eGFP and mRFP1 are linked with amino-acid chains of various lengths (7, 19 and 32 amino acids) shows the two methodologies to be highly correlated. We validate our approach using a small-scale inhibitor screen of a Cdc42 FRET biosensor probe expressed in epidermoid cancer cells (A431) in a 96 microwell-plate format. We also show that acceptor fluorescence anisotropy can be used to measure variations in hetero-FRET in protein-protein interactions. We demonstrate this using a screen of inhibitors of internalization of the transmembrane receptor, CXCR4. These assays enable us to demonstrate all the capabilities of the instrument, image processing and analytical techniques that have been developed. Direct correlation between acceptor anisotropy and donor FLIM is observed for FRET assays, providing an opportunity to rapidly screen proteins, interacting on the nano-meter scale, using wide-field imaging.

Dataset Information

High-performance binary protein interaction screening in a microfluidic format.

Publications

High-performance binary protein interaction screening in a microfluidic format.

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